Parkinson’s disease (PD) is a neurodegenerative disease that is currently not adequately treated with pharmacological approaches and gene therapy is becoming a potential treatment option. Previous studies have shown that the glial cell-derived neurotrophic factor (GDNF) supports the function and survival of nigral dopaminergic neurons, which are the ones decreased in Parkinson’s patients, causing the motor symptoms that characterize this disease. The purpose of this research was to test the transfection efficiency of a human GDNF (hGDNF) containing plasmid in Chinese Hamster Ovary (CHO) and Henrietta Lacks cervical cancer (HeLa) cells and to measure the expression levels post-transfection via qPCR. The data shows lower hGDNF amplification cycle threshold (Ct) values in the transfected samples than in the controls, and thus a statistically significant upregulation of hGDNF relative to normalized expression levels. These findings contribute to the scientific efforts to find a permanent cure for PD.
Matrix-assisted Laser Desorption Ionization-Time of Flight Mass Spectroscopy (MALDITOFMS) has been used routinely over the past decade in clinical microbiology laboratories to rapidly characterize diverse microorganisms of medical importance both at the genus and species levels. Currently, there is keen interest in applying MALDI-TOF MS at taxonomic levels beyond species and to characterize environmental isolates. We constructed a model system consisting of 19 isolates of Deinococcus aquaticus obtained from biofilm communities indigenous to diverse substrates (concrete, leaf tissue, metal, and wood) in the Fox River – Lake Winnebago system of Wisconsin to: (1) develop rapid sample preparation methods that produce high quality, reproducible MALDI-TOF spectra and (2) compare the performance of MALDI-TOF MS-based profiling to common DNA-based approaches including 16S rRNA sequencing and genomic diversity by BOX-A1R fingerprinting. Our results suggest that MALDI-TOF MS can be used to rapidly and reproducibly characterize environmental isolates of D. aquaticus at the subpopulation level. MALDI-TOF MS provided higher taxonomic resolution than either 16S rRNA gene sequence analysis or BOX-A1R fingerprinting. Spectra contained features that appeared to permit characterization of isolates into two co-occurring subpopulations. However, reliable strain-level performance required rigorous and systematic standardization of culture conditions and sample preparation. Our work suggests that MALDI-TOF MS offers promise as a rapid, reproducible, and high-resolution approach to characterize environmental isolates of members of the genus Deinococcus. Future work will focus upon application of methods described here to additional members of this ecologically diverse and ubiquitous genus.
Endolysin p75 is a lysozyme like enzyme produced intracellularly by Enterobacteria phage λ; it hydrolyses β1,4 glycosidic linkages of bacterial peptidoglycan cell walls with Escherichia coli as its targeted host 1 . The objective of this study was to test the scope of antimicrobial activity of λ endolysin p75 in the presence and absence of an outer membrane permeability agent, EDTA. The Nucleotide database in NCBI was used to identify the λ Phage “R” gene that encodes endolysin p75. This gene was PCR amplified and cloned into the expression system Escherichia cloni 10G (Lucigen), a genetically modified E. coli , using a pRham C His Kan Vector with C terminal His tags 2 that allowed for the purification of endolysin p75 protein by affinity chromatography. Single plate serial dilution spotting (SP SDS) plating 3 technique was utilized to quantify the number of colonies formed after exposure to purified endolysin p75 with and without EDTA. Although E. coli was the major focus of the study, the gram negative species Pseudomonas fluorescens and gram positive species Deinococcus radiodurans were also tested to determine if endolysin p75 had a broad spectrum of lysis. Colony forming unit (CFU) data were analyzed with a One way ANOVA ( P ≤ 0.05) to test the statistical significance of antimicrobial activity. Our findings indicate that endolysin p75 causes a significant reduction in CFU’s of gram negative cells only when pre treated with EDTA. As anticipated, the enzyme had no effect on the viability of gram positive cells. Most importantly, this study establishes that cloned endolysin p75 can induce cell lysis from an extracellular direction.
Human immunodeficiency virus type 1 (HIV 1 encodes and employs the use of reverse transcriptase, integrase and protease ( to infect a host cell PR is currently the target of nine FDA approved protease inhibitors ( However, mutant strains of PR have been clinically isolated showcasing resistance to PI treatment The aim of this study was to investigate the trans proteolysis mechanism and drug resistance between mutant PR in relation to the wild type wt form when treated with variable concentrations of darunavir (and tipranavir (To investigate, a previously established cell based assay was used and coupled with engineered enzyme and substrate precursors This was done to specifically amplify the trans proteolysis reactions in transfected mammalian cells by western blotting SDS PAGE with gradient gels were used with sequential antibody probing for detection of the full length substrate (cleavage products (and PR Through these investigations, it was found that the detection profiles generated by the mature PR constructs do not completely reflect drug resistance The fold changes calculated, in respect to the PR WT, each PR mutant equally aligned and misaligned with the reported fold change values (et al 2013 Due to the cytotoxicity of TPV, IC 50 values and fold changes were determined to be inaccurate in some of the detection profiles generated PR MJ 4 mutant 1556 and mutant 634 showed no detection when treated with DRV, suggesting their autoprocessing is resistant to PI treatment PR MJ 4 mutant 4307 mutant 1556 and mutant 634 showed no PR detection when treated with TPV, also suggesting their autoprocessing is resistant to PI treatment. It can be concluded that future assays should not rely solely on mature PR detection but also altered PR precursors as an example of improving drug resistance assessment parameters.
This poster session addresses the research question "What amount of species level variation occurs within Deinococcus aquaticus isolated from the same biofilm habitat and can we resolve it using MALDI-ToF MS?"
Convolutional Neural Networks (CNNs) have been applied to a wide-rage of problems in recent years1,7. In simple terms, these algorithms can be thought of as curve-fitting schemes, designed to take some input (an unknown curve) and generate an appropriate mathematical model for it. With the model, a computer would then be able to predict the output of future curve inputs. CNNs can be broadly viewed as extending the curve-fitting strategy to many more dimensions1,7. MALDI-TOF (Matrix-assisted laser desorption/ionization – Time of Flight) mass spectrometry is a spectroscopic technique commonly used in the analysis of whole cell protein extracts2,5. MALDI-TOF has been shown to reliably produce summary spectra of bacterial proteins resulting in a characteristic fingerprint for a given species2,5. In this investigation, we use a CNN algorithm written in Python (Computer Language) to model changes in MALDI-TOF spectra with respect to D. aquaticus strains in an effort to arrive at a model for predicting the identity an unknown strain using its MALDITOF spectrum.
MALDI-TOF Mass Spectrometry (MS) of bacterial cell protein extracts has been shown to produce a summary spectrum and a characteristic proteomic fingerprint for a given organism 1 . In the present study we used MALDI TOF MS data to derive phylogenetic relationships between nine representatives of the bacterial genus Deinococcus 2 namely; D . aquaticus, D. caeni, D. deserti, D. geothermalis, D. gobiensis, D. grandis, D. indicus, D. misasensis, and D. sonorensis as well as two outlier species, Pseudomonas fluorescens and Vibrio fischeri. This analysis was evaluated against a dendrogram derived from 16S rRNA gene sequencing and yielded congruent findings. However, the MALDI TOF MS approach is rapid, economic and technically straight forward and as such recommends itself as a phylogenetic tool when contrasted with traditional 16S rRNA gene sequencing.
Matrix assisted laser desorption/ionization Time of Flight (MALDI TOF) mass spectrometry of whole cell protein extracts has been shown to produce a summary spectrum and a characteristic fingerprint for a given organism 1 . We have employed a derivative technology to study bacterial phylogenetic relationships known as Protein Extraction Mass Spectrometry (PEMS) and coupled this with genomic information to better understand subpopulation variation. We have recently used this technique to quickly and reliably provide phylogenetic relationships between five species of Deinococcus 2. For the PEMS method, ethanol inactivated cells from 48 hour cultures were protein extracted with 70% formic acid and acetonitrile and samples were spotted in triplicate on the MALDI TOF 3 . Spectra were analyzed and phylogenetic relationships derived using Bionumerics software (http://www.appliedmaths.com/). PEMS was used to elucidate phylogenetic relationships between 21
D. aquaticus isolates obtained from diverse biofilm samples from man made and natural surfaces within the Fox River watershed of northeastern Wisconsin. D. aquaticus is a recently described species with a wide distribution in freshwater habitats. Members of the clade are readily isolated from biofilms growing on diverse substrates. The MALDI TOF spectra analysis of whole cell protein extracts of strains isolated from the same habitat showed similar profiles, but with sufficient cross habitat variation to recognize strain differences. Strain classification with PEMS allowed for a more sensitive characterization of strains as compared to 16S rRNA and house keeping gene analysis as well as genomic fingerprinting by BOX AR1 PCR 4 . In conclusion, the PEMS method used in conjunction with genomic techniques allows for conclusions to be made about ecotypes and the adaptive benefits of differential protein expression in related biofilm habitats.
This study examined molecular profiles of 20 Deinococccus aquaticus isolates from biofilm samples obtained from man made and natural surfaces within the Fox River watershed of northeastern Wisconsin. D. aquaticus is a recently described species with a wide distribution in freshwater habitats. Strains are readily isolated from biofilms growing on diverse substrates. The overall objective of this investigation is to utilize proteomic and genomic techniques to make conclusions about bacterial ecotypes and the adaptive benefits of differential protein expression in related biofilm habitats. MALDI TOF mass spectrometry of whole cell protein extracts has been shown to produce a characteristic spectral fingerprint for a given organism. We employed a derivative technology known as Protein Extraction Mass Spectrometry (PEMS) and coupled this with nucleic acid sequence data from Box A1R PCR, house keeping gene and 16S rDNA analyses to provide a comprehensive profile of subpopulation variation. In conclusion, PEMS, Box A1R PCR, and house keeping gene analyses provided strain resolution. However, the PEMS technique provided the most sensitive characterization of strains. The MALDI TOF spectra analysis of whole cell protein extracts of strains isolated from the same habitat showed similar profiles, but with sufficient cross habitat variation to recognize strain differences.
Numerous species of bacteria communicate by intermolecular signaling mechanisms known as quorum sensing. Quorum sensing induces the regulation of gene expression and cellular function in neighboring cells. Such genes are largely associated with virulence and are involved in processes such as biofilm formation and antibiotic production. Quorum sensing is a density dependent process controlled by the local increase in concentration of N-acyl homoserine lactones (AHL) in the environment. Various quorum-quenching enzymes have been discovered that interfere, or inhibit communication in gramnegative bacteria. The bacterium Deinococcus radiodurans produces two types of quorum-quenching enzymes (QSI), AHL-lactonases and AHLacylases (Koch et al, 2014) that either degrade or bind to AHL rendering the molecule inactive. This has a net effect of altering the regulation of gene expression of bacterial species in the surrounding environment. The objective of the present study is twofold. The first is to characterize the QSI enzymes of D. radiodurans by cloning and expressing the lactonase and acylase genes in E. coli. The second is to study the effect of the proteins on another quorum sensing induced phenomenon, Vibrio fisheri bioluminescence. A modulation of V. fisheri bioluminescence may suggest that QSI enzymes broadly effect virulence gene expression in a range of gram-negative species.
DNA methylation is an epigenetic modification that can alter gene expression without changing the underlying nucleotide sequence. 5-methylcytosine is formed by the binding of a methyl group to the cytosine base of CpG dinucleotides. These modifications may inhibit gene transcription, particularly when present at transcription start sites. Differential DNA methylation has been associated with many human diseases including Alzheimer’s disease (AD; Bradley-Whitman & Lovell, 2013; Coppieters et al., 2013; De Jager et al., 2014; Lunnon et al., 2014; Mastroeni et al., 2010; Van den Hove et al., 2013) although the exact role of epigenetic modifications in the pathology of these diseases has yet to be elucidated. In this study DNA methylation was assayed with an Illumina 450K microarray containing samples from the middle temporal gyrus (MTG) and cerebellum (CBL) of human post-mortem brain. 121 female and 99 male participants with AD neuropathology (n= 220) and 75 female and 98 male participants without AD neuropathology (n= 173), were represented on the microarray, with each subject contributing a MTG and a CBL sample. Raw data was preprocessed and normalized in the R programming platform employing the Minfi package from Bioconductor (Aryee et al., 2014). Local (DMP) and regional (DMR) methylation was detected using the Minfi peak analysis and bumphunter algorithms, respectively. Significant DMPs were determined by false discovery rate qvalue cutoff 0.05 and significant DMRs by family wise error rate cutoff of 0.05. Significant DMRs and DMPs were determined statistically in terms of brain region, disease state, and gender. The biological relevance of each significant DMP and DMR was determined using the UCSC Genome Browser. DMR analysis revealed 137 CpGs to be significantly associated with disease state, 75 of which were specific to the CBL, and 62 to the MTG. Of the 137 CpGs, 42 were found to be significantly correlated with disease in both the CBL and MTG. Statistical analysis of DMRs determined 93 CpG sites were affected by gender state, 53 of which were specific to the MTG and 40 to the CBL. 20 CpGs were significantly correlated with gender in both the CBL and MTG. Five DMRs were found to be significantly correlated with disease, an additional 15 DMRs were significant with respect to gender and disease, and seven additional DMRs were significantly associated with brain region, gender, and disease. Genes found to correspond to disease-associated DMRs include the previously reported AD gene, HLA-DRB5 (Yu et al., 2015), as well as CCDC140, TLX3, HLA-DQA1, ZFP57, ZNF596, RPH3AL and OTX2. This study has described changes in the DNA methylation state of CBL and MTG of participants with AD, determined statistical significance with respect to brain region, disease, and gender, and utilized this information to make novel contributions to the growing list of AD epigenetic signatures.
Bacteria display a type of intercellular molecular communication known as quorum sensing. Quorum sensing induces specific cellular functions in neighboring cells, including the regulation of gene expression and behavior. In Vibrio fisheri (V. fisheri), quorum sensing controls one such behavior, bioluminescence, the ability of an organism to produce light. Individual cells secrete the bacterial quorum-sensing signal, N-acyl homoserine lactone (AHL) into the environment. At high concentrations this molecule binds to the LuxR transcriptional regulator, inducing luminescence. The algae Chlamydomonas reinhardtii (C. reinhardtii), as well as other eukaryotic organisms, produce compounds that mimic AHL (Rajamani et al, 2011). When these compounds are secreted they mimic the signals that are used by bacterial cells and alter or block quorum sensing regulated gene expression (Rajamani et al,2011). The objective of this project is to investigate the AHL signal mimicking ability of compounds secreted by C. reinhardtii by measuring the reduction of bioluminescence of V. fisheri. It is believed that the AHL receptor of V. fisheri is susceptible to inactivation by these secreted compounds, thereby inhibiting density specific cellular functions, such as bioluminescence and biofilm formation. This ability to inactivate quorum sensing suggests that C. reinhardtii secretes compounds with a conserved lactone ring structure that may act as pathway antagonists. The resultant inhibition of quorum sensing may ultimately be usefully employed to disrupt the activities of pathogenic biofilm forming bacteria.
Members of the genus Deinococcus are able to withstand extreme radiation, extended periods of desiccation, and oxidative stress. The ability of the bacteria to withstand the damage incurred is attributed, in part, to a DNA repair mechanism that is highly conserved across the genus. Within hours after high doses of radiation, it has been shown that D. radiodurans can repair hundreds of double stranded breaks in its DNA—a unique characteristic of the taxon. Several genes are involved in the DNA repair system but IrrE (also known as PprI) seems essential for the activation of critical repair genes. Recently the mechanism underlying this activation has been described in D. deserti. In this species, once activated, IrrE cleaves a repressor protein, DdrO, allowing for the expression of a number of genes that share a common upstream regulatory motif (Ludanyi et al., 2014). Unlike IrrE, the protein DdrO appears not to be as widely conserved across the Deinococci. The present study aims to extend these findings into other Deinococci species. The cloning of irrE and ddrO from D. radiodurans and D. hopiensis into E. coli allows us to compare the DNA repair pathway in these species to that described for D.deserti.
It is anticipated that future atmospheric warming will increase the salinity of soils, inducing greater plant oxidative stress and poorer crop yields. In this scenario the recombinant introduction of superior oxidative stress defense mechanisms may be considered advantageous. Furthermore the engineering of algae to be grown in brackish open pond systems may provide a defense against opportunistic air borne organisms. In this case also, the heterologous expression of superlative antioxidative enzymes may be a preferred strategy. The bacterial genus Deinococcus may well be a productive source of these high-caliber enzymes. Deinococcus is well-known for its adaptation to extreme environments. These organisms are highly resistant to desiccation, oxidative challenges and ionizing and non-ionizing radiation. Deinococcus radiodurans has been the most studied and best understood member of the genus since its discovery from gamma irradiated canned meat in 1956. The resilience of D. radiodurans has been attributed to multiple factors such as multiple redundancies of its genome and metabolic pathways, an ability to accurately repair massive damage to its DNA, and highly effective mechanisms to reduce the detrimental effects of reactive oxygen species (ROS). ROS are created naturally as a side product of aerobic respiration. Aerobic organisms produce several enzymes, most notably catalase, to remove ROS from cells. The catalase of D. radiodurans has significantly higher activity than that found in most other organisms and is a major factor in the ability of D. radiodurans to survive long periods of desiccation and oxidative stress. The goal of this project is to further characterize the activity of this enzyme in vivo under conditions of salt exposure.
The pentose phosphate pathway (PPP) is a ubiquitous metabolic pathway that parallels glycolysis and is the primary pathway responsible for the generation of NADPH and ribose 6-phosphate1. Glucose 6-phosphate dehydrogenase (G6PD) is the rate limiting enzyme of the oxidative phase of the PPP and is responsible for the reduction of NADP+ through the oxidation of glucose 6-phosphate2. NADPH is required for the synthesis of fatty acids and for the generation of antioxidants that scavenge reactive oxygen species (ROS) within cells2. The extremophiles Deinococcus radiodurans and Deinococcus geothermalis have been previously linked to increased G6PD activity and concentrations of NADPH in comparison to Escherichia coli as they require additional cellular protection for combating ROS and increased ribose production for DNA repair3,4. In this study, the reduction of NADP+ was assayed to measure G6PD activity in seven species of Deinococcus whose metabolic preferences have yet to be characterized. The specific activity (U/mg) of G6PD was then compared with the ability of a species to withstand UV exposure and oxidative challenge5. Our findings indicate that a higher specific activity correlates with resistance in D. radiodurans, D. metalli, and D. grandis, whereas a lower G6PD specific activity is found in the less resistant species D. pimensis, D hopiensis, D. caeni, and D. indicus.
The prokaryotic genus, Deinococci is known for its ruggedness. Members of this genus are commonly resistant to extreme levels of ionizing radiation, oxidative stress and dessication. The Deinococci synthesize carotenoids, forty carbon, organic pigments and as a class these are known to be potent antioxidants. The Deinococcus carotenoid most studied to date is deinoxanthin, derived from the well-studied species D. radiodurans. It may well be that the effectiveness of the Deinococcus in quenching reactive oxygen species and combating oxidative stress in general is derived from their synthesis of carotenoids. This study looks to isolate carotenoids from D. hopiensis, a more recently discovered and thus less characterized member of the genus, and compare those carotenoids with deinoxanthin.
Numerous microorganisms have been harnessed for their ability to detoxify or immobilize a variety of pollutants. However, most of these microorganisms are sensitive to the damaging effects of ionizing radiation. This limits the applicability of these organisms for bioremediation. Some of the most radiation resistant organisms yet discovered exist within the genus Deinococcus. For instance, D. radiodurans is a nonpathogenic, solvent tolerant soil bacterium that can grow continuously in the presence of high levels of ionizing radiation with little or no effect on its growth rate. It can similarly withstand desiccation and oxidizing agents. These are characteristics found across the Deinococcus genus which makes the group as a whole suitable for a host of bioremediation challenges. However, an ideal bioremediation organism should also have a well characterized extracellular proteome. Although the extracellular proteome of D. radiodurans has been extensively studied, much less is known about the secreted proteins of the 47 or so other species within the genus. The present study looks at secreted proteases and glycosyl hydrolases (amylases) from two other Deinococci: D. gobiensi, D. deserti and D. hopiensis and compares them to the best know representative of the genus, D. radiodurans.
Studies have shown that mutations in eight splicing factor genes (SF 3 B 1 U 2 AF 1 SRSF 2 ZRSR 2 SF 3 A 1 PRPF 40 B, U 2 AF 2 and SF 1 are associated with myelodysplastic syndrome ( and acute myeloid leukemia ( The goal of this study is to understand the mechanism underlying this association due to mutations in the SF 3 A 1 gene. The SF 3 A 1 gene encodes for one of three subunits of the human splicing factor SF 3 a, which interacts with SF 3 b and the 12 S U 2 small nuclear ribonucleoprotein particle snRNP to form the 17 S U 2 snRNP of the spliceosome complex. The 17 S U 2 snRNA binds to the branch point sequence on the pre mRNA and participates in the removal of introns SF 3 A 1 has previously been shown to interact with stem loop 4 (SL 4 on U 1 snRNA during spliceosome assembly, acting as an important molecular bridge between the 5 and 3 splice sites (Sharma et al 2014 The SL 4 binding site on SF 3 A 1 is not known. This study aims to identify the binding domain in SF 3 A 1 for SL 4 and to observe how mutations in SF 3 A 1 affect SL 4 binding For this we have made C and N terminal deletion clones of SF 3 A 1 to express 6 x His tagged proteins that will be used for RNA binding analysis using UV crosslinking and electrophoretic mobility shift assay. Identifying mutations that affect binding of SF 3 A 1 to SL 4 will indicate how splicing errors contribute to MDS and AML diseases.
Tick-borne relapsing fever (TBRF) is caused by the bacterium, Borrelia hermsii and is transmitted to humans by the bite of an infected Ornithodoros hermsi (soft bodied tick). The purpose of this study was to determine the prevalence of B. hermsii in Barred Owls collected from Hoopa Valley Indian Reservation. DNA was extracted from the blood samples and subsequent qPCR was performed as a method to detect for the presence of B. hermisii. Of the samples analyzed, there were zero positives for B. hermsii.
The approximately 60 species of the genus Deinococcus are typically characterized as radiation-resistant mesophiles although there is much functional and morphological variation within the group. The relationship of Deinococcus to other microbial groups has also been difficult to establish. For instance, Deinococcus was originally placed in the Micrococcus genus (1). Deinococcus is known to share a number of genes with the Archaebacteria but unlike the Archaebacteria has peptidoglycan in its cell wall. Furthermore, they stain positive with gram stain but have a second membrane, characteristic of gram negative bacteria. In short, a systematic phylogenetic analysis of the genus is necessary. To establish phylogenetic relationships it is typical to rely on a comparison of the sequence of the small subunit ribosomal RNA (16S) gene. However, it has been pointed out that a phylogenetic tree inferred using the 16S rDNA gene may differ significantly from a tree based on other marker genes (2). Matrix-assisted laser desorption/ionization – Time of Flight (MALDI-TOF) mass spectrometry of whole cell protein extracts has recently been developed to produce a summary spectrum and a characteristic fingerprint for a given organism (1). While MALDI-TOF spectra has been used to rapidly identify many species of bacteria in a clinical setting, the technique has not been used to phylogenetically fingerprint Deinococcus. This study develops a protocol for whole cell protein extraction and MALDI-TOF analysis to establish phylogenetic relationships between D. radiodurans, D. gobiensis, D. deserti and D. hopiensis.
Characterization of microbial environmental isolates by MALDI-TOF Mass Spectrometry (MS) has been used recently to ascertain taxonomic information at the genus and species levels. Our goal in this study was to investigate whether MALDI-TOF MS affords higher taxonomic resolution when examining proteomic profiles below the species level. Nineteen isolates of Deinococcus aquaticuswere obtained from diverse biofilm habitats in and around the Lake Winnebago –Fox River system in Wisconsin, USA. We developed a methodology to produce rapid and reproducible MALDI-TOF spectra by: 1) design of sample preparation techniques to reduce proteomic background noise and 2) use of comparative integration of multiple DNA profiling methods such as 16S rRNA gene sequencing and BOX-A1R fingerprinting. Our results suggest that MALDI-TOF MS shows greater taxonomic resolution than either 16S rRNA gene sequencing or BOX-A1R fingerprinting. Two separate subgroups were characterized by spectra that contained features that appeared unique to each group. We conclude that MALDI-TOF MS represents a rapid, high resolution, and readily reproducible method for the proteomic characterization of environmental isolates of the Deinococcusgenus. Future work will include application of current methods to additional members of the genus.